Chimeric antagonist anth1

ABSTRACT

A recombinant chimeric antagonist formed by a 60 amino acid fragment of the N-terminal region of human interleukin 2 (IL-2) fused to the N-terminal of the extracellular region of the alpha subunit of the gamma IFN (IFN γ) receptor. In vitro this protein has a T cell growth stimulating activity, it inhibits the growth stimulating activity of IL-2 in T cells, it inhibits the induction of HLA-DR by IFN γ and it inhibits the antiproliferative activity of γ IFN. This invention can be applied in the field of medicine for the treatment of several pathologies such as autoimmune diseases, graft rejections, chronic inflammations, sepsis, ischemia and reperfusion syndrome and atherosclerosis.

This application is a U.S. National Phase Application of InternationalApplication No. PCT/CU03/00006 filed on May 8, 2003. The specificationof International Application No. PCT/CU03/00006 is hereby incorporatedby reference in its entirety.

This application also asserts priority to Cuban Application No.CU2002/0095 filed on May 10, 2002. The specification of CubanApplication No. CU2002/0095 is hereby incorporated by reference in itsentirety.

BACKGROUND OF THE INVENTION

The present invention is related to biological science, biotechnologyand medical science, in particular with a drug that may inhibit thebiological activities of interleukin-2, (IL-2) and gamma Interferon (IFNγ), two cytokines that act in the regulation of several functions of thebody, that show increased amounts when found in a pathological state.This in turn avoids the inactivation of the immune system that endangersthe lives of patients.

The production of cytokines by T helper lymphocytes (CD4+) and bycytotoxic T lymphocytes (CD8+) generate a pattern of cytokine productionthat have been identified as Th1 and Th2. The Th1 pattern ischaracterised by the production of IL-2, a tumor necrosis alpha (TNF α)and IFN γ, while Th2 pattern responds to the production of IL-4, IL-5,IL-6 and others. This type of response plays an important role in thebody protection, as well as being the promoters of differentimmunopathological reactions.

There are a series of situations in which the inflammatory anduncontrolled immune reactions lead to the presence, development andperpetuation of inflammatory and autoimmune diseases. There aredemonstrated examples on the pathological roles of the IL-2 and IFN γ insome of these diseases.

Multiple sclerosis is a degenerative demyelenating autoimmune disease.The role of IFNγ is very clear for this autoimmune disorder. Hence, ithas been demonstrated in a clinical trial using IFNγ that this treatmentproduces the exarcebation of the disease (Panitch H S. et al.Exacerbations of multiple sclerosis in patients treated with interferongamma. Lancet 1, 893-5, 1987.). It has also been demonstrated that thelevels of messenger RNA, as well as the protein corresponding to IL-2and IFNγ are found to be high in patients suffering from this disease(Lin J. et al. IL-2, IFN-gamma, and TNF-alpha mRNA expression inperipheral blood mononuclear cells in patients with multiple sclerosis.Chung Kuo I Hsueh Ko Hsueh Yuan Hsueh Page 19, 24-8, 1997). Theproduction of these two cytokines by patient's cells has suggested theiruse as markers of the relapse of multiple sclerosis (Philippe J. et al.In vitro TNF-alpha, IL-2 and IFN-gamma production as markers of relapsesin multiple sclerosis. Clin Neurol Neurosurg 98, 286-90, 1996). It hasalso been observed that the IL-2 and IFNγ are involved in the activationof non-specific lymphocytes that lead to the demyelination of thecentral nervous system (Martino G. et al. Proinflammatory cytokinesregulate antigen-independent T-cell activation by two separatecalcium-signaling pathways in multiple sclerosis patients. Ann Neurol43, 340-49, 1998). A clinical trial is being carried out using ananti-IL-2 antibody known as Daclizumab in patients with this diseasethat do not respond to therapy with β IFN.

Lupus erythematosus is another systemic autoimmune disease where thepresence of high levels of IL-2 and IFNγ have been associated toexacerbations of this disease (Viallard J F. et al. Th1 (IL-2,interferon-gamma (IFN-gamma)) and Th2 (IL-10, I cytokine production byperipheral blood mononuclear cells (PBMC) from patients with systemiclupus erythematosus (SLE). Clin Exp Immunol 115, 189-95, 1999). On theother hand, the absence of the receptor for IFNγ decreases theproduction of autoantibodies in lupus animal models (Haas C. et al.IFN-gamma receptor deletion prevents autoantibody production andglomerulonephritis in lupus-prone (NZB×NZW)F1 mice. J Immunol 160,3713-18, 1998) and the presence of the soluble receptor for IFNγinhibits the presence of the disease (Ozmen L. et al. Experimentaltherapy of systemic lupus erythematosus: the treatment of NZB/W micewith mouse soluble interferon (receptor inhibits the onset ofglomerunephritis. Eur J Immunol. 25, 6-12, 1995). This was recentlytested using a murine lupus model with a chimeric protein containing theextracellular region of the IFNγ fused to the Fc fraction of theimmunoglobulins (Lawson B R. et al. Treatment of murine lupus with cDNAencoding IFN-gammaR/Fc. J Clin Invest. 106, 207-15, 2000). The efficacy,however of this type of molecule in lupus erythematosus could be limitedby a demonstrated dysfunction of the Fc receptor in patients with lupuserythematosus (Frank M M. et al. Defective reticuloendothelial systemFc-receptor function in systemic lupus erythematosus. N Engl J Med 300,518-23, 1979, Dijstelbloem H M. et al. Fcgamma receptor polymorphisms insystemic lupus erythematosus: association with disease and in vivoclearance of immune complexes. Arthritis Rheum 43, 2793-800, 2000).

Myastenia gravis is considered an organ-specific autoimmune diseasemediated by anti-acetyl coline receptor autoantibodies and dependent onT cells, characterized by muscular weakness and fatigue. It was recentlydemonstrated that the IFNγ favors the formation of autoantibodiesagainst the acetyl coline receptor; while the absence of the IFNγreceptor decreases the susceptibility to the disease in animal models(Zhang G X. et al. Mice with IFN-gamma receptor deficiency are lesssusceptible to experimental autoimmune myasthenia gravis. J Immunol 162,3775-81, 1999). The IL-2 and other cytokines contribute, together withthe IFNγ to the development of the disease (Zhang G X. et al. Cytokinesand the pathogenesis of myasthenia gravis. Muscle Nerve. 20, 543-51,1997).

In type 1 Diabetes (insulin dependent) or Diabetes mellitus, the betacells of the pancreas are destroyed by an autoimmune mechanism. There isin vitro evidence that the IFNγ may be toxic for the beta cells of thepancreas (Sternesjo J. et al. Effects of prolonged exposure in vitro tointerferon gamma and tumor necrosis factor-alpha on nitric oxide andinsulin production of rat pancreatic islets. Autoimmunity 20, 185-90,1995, Dunger A. et al. Tumor necrosis factor-alpha and interferon-gammainhibit insulin secretion and cause DNA damage in unweaned-rat islets.Extent of nitric oxide involvement. Diabetes 45, 183-9, 1996, Baldeon ME. et al. Interferon-gamma independently activates the MHC class Iantigen processing pathway and diminishes glucose responsiveness inpancreatic beta-cell lines. Diabetes 46, 770-8, 1997). However, otherstudies demonstrate that the action of the IFNγ on the cells producinginsuline in the pancrease is indirect (Sarventick N. et al, Loss ofpancreatic islet tolerance induced by beta-cell expression ofinterferon-gamma. Nature, 346, 844-7, 1990). Most probably, its actionis activated through macrophages for the production of IL-1, TNFα andnitric oxide that do act directly on the beta cells and stimulate theexpression of MHC I in beta cells, thus favoring their destruction bycytotoxic lymphocytes (Thomas H E. et al. IFN-gamma action on pancreaticbeta cells causes class I MHC upregulation but not diabetes. J ClinInvest, 102, 1249-57, 1998, Thomas H E et al. Beta cell destruction inthe development of autoimmune diabetes in the non-obese diabetic (NOD)mouse. Diabetes Metab Res Rev 16, 251-61, 2000). It has also been shownthat the absence of the IFNγ delays the appearance of diabetes, althoughit does not prevent it (Hultgren B. et al. Genetic absence ofgamma-interferon delays but does not prevent diabetes in NOD mice.Diabetes 45, 812-7, 1996). Several reports show how the inactivation ofthe biological activity of IFNγ may be used to prevent diabetes(Debray-Scahs M. et al. Prevention of diabetes in NOD mice treated withantibody to murine IFN gamma. J Autoimmun 4, 237-48, 1991, Moosmayer D.et al. A bivalent immunoadhesin of the human interferon-gamma receptoris an effective inhibitor of IFN-gamma activity. J Interferon CytokineRes 15, 1111-5, 1995, Prud'homme G J. et al. Prevention of autoimmunediabetes by intramuscular gene therapy with a nonviral vector encodingan interferon-gamma receptor/IgG1 fusion protein. Gene Ther 6, 771-7,1999). It has also been seen that IL-2 as an activator of T lymphocytescan contribute to this reaction in the destruction of cells producinginsulin. A clinical trial was recently started for the treatment of type1 diabetes in children/adolescents of between 10 and 21 years of agewith a recent diagnosis of the disease using the antibody anti IL-2Daclizumab (Riley Hospital for Children. Project: Prevention of DiabetesProgression Trial (PDPT). www.rileyhospital.org.). This study has beendesigned to prevent the progression of the destruction of beta cells inrecently diagnosed children.

The rejection to grafts is a complex process in which the cell mediatedimmunity and the circulating antibodies play an important role. Thestandard anti-rejection therapies use combinations of drugs such ascyclosporin, rapamycin, azatioprin, steroids and others. However, evenwith this therapy, more than 50% of the persons receiving the kidneysslowly reject their graft in 10 years. The graft disease against thehost is the main cause of death among patients grafted with bone marrow.In these reactions that prevent grafts and compromises the lives of thegrafted patients it has been demonstrated that both the IL-2 and theIFNγ contribute to their development (Hu H Z. et al. Kinetics ofinterferon-gamma secretion and its regulatory factors in the early phaseof acute graft-versus-host disease. Immunology 98, 379-85, 1999,Nakamura H. et al. Serum levels of soluble IL-2 receptor, IL-12, IL-18,and IFN-gamma in patients with acute graft-versus-host disease afterallogeneic bone marrow transplantation. J Allergy Clin Immunol. 106,S45-50. 2000).

Reumatoide arthritis (RA) is a chronic systemic disorder of unknownethiology, characterized by inflammation, sinovial hyperplasia and thedestruction of the affected joints. IL-2 is generally considered apro-inflammatory citokine that exacerbates the state of type Th1diseases such as autoimmune arthritis. Recent studies have shown thatthe IL-2 messenger RNA is increasing during the acute phase of thearthritis induced by a collagen in an animal model (Thornton S. et al.Heterogeneous effects of IL-2 on collagen-induced arthritis. J Immunol165, 1557-63, 2000). On the other hand, the exacerbation of the diseasehas been found in animal models to be associated to IFNγ increase(Tellander A C. et al. Potent adjuvant effect by anti-CD40 incollagen-induced arthritis. Enhanced disease is accompanied by increasedproduction of collagen type-II reactive IgG2a and IFN-gamma. J Autoimmun14, 295-302, 2000). Both the IL-2 and the IFNγ have been significantlyincreased in the sinovial tissue of patients with RA (Canete J D et al.Differential Th1/Th2 cytokine patterns in chronic arthritis: interferongamma is highly expressed in synovium of rheumatoid arthritis comparedwith seronegative spondyloarthropathies. Ann Rheum Dis 59, 263-8, 2000).

The inflammatory disease of the intestine consists of twogastrointestinal disorders: Crohn disease and ulcerative colitis. Thesediseases are characterized by the chronic inflammation of the intestine.Crohn disease is an inflammatory disorder that is extended around theinternal line of the intestinal wall and penetrates in its deepestlayers. This inflammation can be found in any part of the digestivesystem (esophagus, stomach, small intestine, large intestine or theanus). The Protein Desig Labs company has announced the start of phaseI/II clinical trials in patients with moderate to severe crohn diseaseusing an anti IFNγ antibody (SMART Anti-Gamma Interferon Antibody)(Fremont, Calif. Protein Design Labs Announces Phase I/II Trial ofSMART″ Anti-Gamma Interferon Antibody in Crohn's Disease. Protein DesignLabs, Inc. (Nasdaq), Jan. 10, 2001). Ulcerative colitis is confined tothe mucose and sub-mucose of the large intestine (the colon or rectus).Recently, in the Annual Congress of the American Association ofGastroenterology it was shown how the decrease in the levels of IFNγ inthe bloodstream is a remission marker in a mouse model for colitis(Yaron I. Annual meeting of the American Gastroenterology Association.May 20-23, 2001. Georgia World Congress Center. Atlanta, Ga.).

Septic shock is the result of the dissemination of microorganisms ofrevere infections through the bloodstream. This is more frequentlyproduced by Gram-negative bacilli acquired at the hospitals and it ismore common in immunocompromised patients and those having chronicdiseases. In ⅓ of the patients it is produced by Gram-positive germs andby Candida albicans. Both in the septic shock produced by the gramnegative and the Gram positive bacteria, the IFNγ and the IL-2contribute to the lethality of the inflammatory reactions in which theyparticipate. The IFNγ is a lethal mediator in animal models with septicshock (Heremans H. et al. Interferon gamma, a mediator of lethallipopolysaccharide-induced Shwartzman-like shock reactions in mice. JExp Med 171, 1853-69, 1990, Wysocka M. et al. Interleukin-12 is requiredfor interferon-gamma production and lethality inlipopolysaccharide-induced shock in mice. Eur J Immunol 25, 672-6, 1995,Kuschnaroff L M et al. Increased mortality and impaired clonal deletionafter staphylococcal enterotoxin B injection in old mice: relation tocytokines and nitric oxide production. Scand J Immunol 469-78, 1997). Asis the case for animal models of other diseases, the absence of IFNγreceptor in these animals make them resistant to endotoxic shock (Car BD et al. Interferon gamma receptor deficient mice are resistant toendotoxic shock. Exp Med 179, 1437-44, 1994). Similarly, several reportsshow the participation of IL-2 in the development and lethality ofseptic shock (Micusan V V, et al. Production of human and murineinterleukin-2 by toxic shock syndrome toxin-1. Immunology 58, 203-8,1986, Arad G, et al. Superantigen antagonist protects against lethalshock and defines a new domain for T-cell activation. Nat Med. 6, 378-9,2000, Stevens D L. et al. Streptococcal toxic shock syndrome associatedwith necrotizing fasciitis. Annu Rev Med 51, 271-88, 2000). Mononuclearcells grown in vitro with IL-2 segregate secondary cytokines such asIL-1, TNFα and IFNγ are implicated in the pathophysiology of septicshock.

Vulgar psoriasis is a complex and multigenic skin disease that ispotentially mediated by pro-inflammatory cytokines produced by damaged Tcells. An unappropriate chronic expression of these cytokines leads tothe immune activation of cells and to tissue damage. This ischaracterised by an excessive production of skin cells and thegeneration of blood vessels that are probably responsible for theredness and plate formation that are part of this disease. Thepathological role of IFNγ and IL-2 has been evidenced for psoriasis.Most of the epidermal cells in vulgar psoriasis produce IL-2, IFNγ andTNFα that are defined as T cytotoxic cells. High levels of IFNγ and IL-2and not of IL-4 have been detected in psoriasis patients. This may berelated to disbalance in T cell populations that contribute to asustained or chronic immune activation of these cells (Schaak J F et al.T cells involved in psoriasis vulgaris belong to the Th1 subset. JInvest Dermatol 102, 145-9, 1994, Austin L M, et al. The majority ofepidermal T cells in Psoriasis vulgaris lesions can produce type 1cytokines, interferon-gamma, interleukin-2, and tumor necrosisfactor-alpha, defining Tc1 (cytotoxic T lymphocyte) and TH1 effectorpopulations: a type 1 differentiation bias is also measured incirculating blood T cells in psoriatic patients. J Invest Dermatol 113,752-9, 1999). An encouraging result using Daclizumab has been showingfor psoriasis treatment (Krueger J M et al. Successful in vivo blockadeof CD25 (hihg-affinity interleukin 2 receptor) on T cells byadministration of humanized anti-Tac antibody to patients withpsoriasis. J Am. Acad. Dermatol. 43, 448-58, 2000). Recently a new trialhas been started with this drug (Fremont, Calif. Protein Design LabsPresents Three Humanized Antibodies in Clinical Development forPsoriasis at International Psoriasis Symposium. Jun. 22, 2000. ProteinDesign Labs, Inc. (PDL) Nasdaq).

There are other less studied diseases where the use of an antagonistagainst the IL-2 and the IFNγ can also be of use; in this case there isatherosclerosis and ischemia/reperfusion. Atherosclerosis andpost-grafting atherosclerosis are characterized by the expansion of thearterial intima as a result of the infiltration of mononuclearleukocytes, the proliferation of vascular smooth muscle cells and theaccumulation of extracellular matrix, as well as the presence of IFNγ(Ross R. Atherosclerosis—an inflamatory disease. N. Engl. J. Med. 340,115-26, 1999, Hansson G K et al. Immune mechanisms in atherosclerosis.Arteriosclerosis 9, 567-78, 1989, y Libby P et al. Functions of vascularwall cells related to development of transplantation-associated coronaryarteriosclerosis. Transplant. Proc. 21, 3677-84, 1989). It has beendemonstrated that the exogenous IFNγ increases the atherosclerosis in ananimal model (Whitman S C et al. Exogenous interferon-gamma enhancesatherosclerosis in apolypoprotein E−/− mice. Am J Pathol 157, 1819-24,2000). On the other hand, it has been demonstrated that theneutralization of IFNγ in the serum, as well as the absence of its gene,decreases the extension of the expansion of the intima (Gupta S et al.IFN γ potentiates atherosclerosis in ApoE knock-out mice. J. Clin.Invest. 99, 2752-61, 1997, Nagano H et al. Interferon γ deficiencyprevents coronary arteriosclerosis but not myocardial rejection intransplanted mouse hearts. J. Clin. Invest. 100, 550-57, 1997,Räisänen-Sokolowski, A. et al. Reduced transplant arteriosclerosis inmurine cardiac allografts placed in interferon γ knockout recipients.Am. J. Pathol. 152, 359-65, 1998). More recently it was proven that theIFNγ promoted atherosclerosis action in the absence of leukocytes(Tellides G et al. Interferon γ elicits arteriosclerosis in the absenceof leukocytes. Nature 403, 207-11, 2000). Ischemia and reperfusion arecharacterized by the interruption of blood flow in an area, with theconsequent elimination of oxygen and nutrient supply and the reperfusionand total or partial restoration of blood flow to the tissue that wasischemic, which is clinically frequent. It can be observed duringhypovolemic and septic shock, myocardium infarct, embolism,compartimental syndrome, freezing, organ graft, etc. Tissue hypoxia, inany case, produces an alteration of the cell metabolism from whichconstantly better-known complex biochemical and molecular modificationsare derived. The damage due to reperfusion consequently producescellular death and endothelial dysfunction produced by the restorationof blood tissue. IFNγ and IL-2 have been reported to be mediators of thedamage produced on the organs through ischemia and reperfusion (SerrickC et al. The early release of interleukin-2, tumor necrosis factor-alphaand interferon-gamma after ischemia reperfusion injury in the lungallograft. Transplantation 58, 1158-62, 1994, Marck A R C et al.Ischemia/Reperfusion-Induced IFN-gamma Up-Regulation: Involvement ofIL-12 and IL-18. The Journal of Immunology 162, 5506-10, 1999). Severalauthors have described antagonists against IFNγ. The inhibition of theantiviral activity of the human IFNγ humano by its recombinant solublereceptor has been described in the European patent EP 0 393 502 A1. Ithas also been described that the recombinant soluble receptor for murineIFNγ inhibits the presence of glomerulonephritis in mice (Ozmen L. etal. Experimental therapy of systemic lupus erythematosus: the treatmentof NZB/W mice with mouse soluble interferon-gamma receptor inhibits theonset of glomerulonephritis. Eur J Immunol. 25, 6-12, 1995). Threemurine IFNγ have been constructed. These consist of chimeric proteinsformed by the extracellular region of the receptor in mice for IFNγ andconstant domains of immunoglobuline molecules. These constructionsneutralize the antiviral activity of the mouse IFNγ and have a prolongedmean life in the blood (Cornelia K. et al. Construction, purification,and characterization of new interferon gamma (IFN γ) inhibitor protein.J. Biol. Chemestry. 267, 9354-60, 1992 and European patent EP 0 614 981A1). The potential use of a fragment of immunoglobulin or its Fc regionfused to the soluble IFNγ receptor to be used in lupus erythematosus inhumans can be limited by a demonstrated dysfunction of the receptor forthe Fc of this autoimmune dysorder (Frank M M et al. Defectivereticuloendothelial system Fc-receptor function in systemic lupuserythematosus. N Engl J Med. 300, 518-23, 1979). This type of inhibitoris monofunctional and it therefore has a smaller scope of action.

In mice, the use of neutralizing anti-IFNγ antibodies decreases theexpressions of the graft disease against the host (Mowat A. et al.Antibodies to IFN gamma prevent immunologically mediated intestinaldamage in murine graft-versus-host reaction. Immunology 68, 18-23,1989). In a skin allograft study, anti-IFNγ antibodies inhibited therejection only if the graft was incompatible with the MHC class IIantigens. This would suggest that the IFNγ constributes to the rejectionof the allograft through the induction of MHC class II antigens(Rosenberg A. et al. Specific prolongation of MHC class II disparateskin allografts by in vivo administration of anti-IFN gamma monoclonalantibody. J. Immunol. 144, 4648-50, 1990). Single strand antibodies witha variable region (scFv) against human IFNγ expressed in bacteria havealso been obtained and proven to be efficient in neutralizing thebiological activity of murine IFNγ (Froyen G. et al. Bacterialexpression of a single-strand antibody fragment (scFv) that neutralizesthe biological activity of human interferon γ. Mol Immunol. 30, 805-12,1993). The use of antibodies in human therapy faces the problem of theresponses of the host against the immunogenic regions of theseheterologous molecules, and in the cases of chimera and humanizedantibodies, to the loss of affinity and specificity (Merluzzi S et al.Humanized antibodies as potential drugs for therapeutic use. Adv ClinPath, 4, 77-85, 2000) as well as to expressions of toxicity (Clark M etal. Antibodies to IFN gamma prevent immunologically mediated intestinaldamage in murine graft-versus-host reaction. Immunology 68, 18-23,2000).

Other analogous solutions have also been described. Mixtures of acytokine with its soluble receptor have been proposed, but its purposewas to potentiate the effect of the cytokines. In this case the cytokineand its receptor are independently produced and later mixed in onepreparation as demonstrated in the American patent WO 94/21282.Cyclosporin A, FK506 and rapamycin are potent suppresors of the immunesystem, especially of T cells, that are used to prevent graft rejection.The first two, inhibit the signal transduction started by the antigenreceptor to T cells that lead to the transcription of the earlyactivation genes. This includes the transcription of the gene thatcodifies for the IL-2 needed for the transition of the state of rest G0to phase G1 of the cell cycle. Rapamycin has no effect of the earlysynthesis of the cytokines by the T cells, but it inhibits the responseof these cells to the IL-2 required for the transition from phase G1 toS of the cell cycle (Waldmann T. A. et al. The IL-2/IL-2 receptorsystem: a target for rational immune intervention. Immunology Today 14,264-70, 1993). Rapamycin allows the specific activation of T cells butavoids their clonal expansion through the interference with signaling bymeans of the beta strand of the IL-2 receptor (RβIL-2) (Woerly G. et al.Effect of rapamycin on the expression of the IL-2 receptor (CD25). ClinExp Immunol 103, 322-7, 1996). Cyclosporin A, has significantlyincreased the survival of renal allografts, while it also decreasesautoimmune diseases. The use of these immunosuppressors has beenlimited, because of their toxic effects that include gastrointestinalcomplications, gum hypertrophia and especially dose dependentnephrotoxicity, and hypertension. (Hortelano S. et al. Potentiation bynitric oxide of cyclosporin A and FK506-induced apoptosis in renalproximal tubule cells. J Am Soc Nephrol 11, 2315-23, 2000, Tsimaratos Met al. Kidney function in cyclosporine-treated paediatric pulmonarytransplant recipients. Transplantation 69, 2055-9, 2000). Monoclonalantibodies that block the interaction of IL-2 with its receptor havebeen used in animal models to inhibit the graft-versus-host disease andthe rejection of allografts. These antibodies were also used in rodentsto suppress autoimmune disorders. In clinical trials, the antibodiesagainst the alpha strand of the IL-2 receptor (RαIL-2) improved thegraft disease against the host that was resistent to steroid treatment.However, these efforts are limited because of the antigenicity of theseantibodies. Single strand antibodies of variable region have been foundto inhibit the union of the IL-2 to the γ subunit of the IL-2 receptorand interfere with the biological activity of the IL-2 tested in themurine cell line CTLL-2 as described in the European patent EP 0 621 338A2. The creation of humanized antibodies that improve this type oftherapy, still have the drawbacks referred to previously. In this typeof therapy monolonal antibodies conjugated with toxins or radioactivelymarked, have also been tested (Waldmann T. A. Genetically engineeredmonoclonal antibodies armed with radionuclides. Year Immunol. 7, 205-12,1993). The use of toxins fused to antibodies anti IL-2 or IL-2 toeliminate cells that express the receptor for IL-2 and contribute to thedevelopment of pathogenical stages have been described as stated in theAmerican patent WO 92/20701 and in the European patent EP 0 369 316 A2.The non-specific toxicity of the immunotoxins (Frankel A. E. et al.Clinical trials of target toxins. Cancer Biology 6, 307-17, 1995) aswell as their immunogenicity (Chen S-Y. et al. Design of geneticimmunotoxin to eliminate toxin immunogenicity. Gene Therapy 2, 116-23,1995) has made the use of these drugs hardly recommendable. Anti IL-2antibodies are being developed for the treatment of RA (Simon L S. etal. New and future drug therapies for rheumatoid arthritis. Rheumatology(Oxford) 39 Suppl 1:36-42, 2000). Antibodies targeted against the RαIL-2are being used to avoid the rejection of kidney grafts (Olyaei A J etal. Use of basiliximab and daclizumab in kidney transplantation. ProgTransplant 11, 33-7, 2001). The limits of the use of antibodies inprolonged therapies have been described above.

The neutralization of pro-inflammatory cytokines such as TNFα (BeutlerB. et al. Passive immunization against cachectin/tumor necrosis factorprotects mice from lethal effect of endotoxin. Science 229, 869-71,1989) or IL-1 (Natanson C. et al. Selected treatment strategies forseptic shock based on proposed mechanism of pathogenesis. Ann. Intern.Med. 120, 771-83, 1994) decrease mortality due to sepsis in severalanimal models. However, in clinical trials using antagonists againstIL-1 (antagonist of the IL-1 receptor) (Fischer C. J. et al. Recombinanthuman interleukin-1 receptor antagonist in the treatment of patientswith sepsis syndrome: results from randomized, double-blind,placebo-controlled trial. JAMA 271, 1836-43, 1994) and against the TNFα(a recombinant chimeric protein: soluble receptor TNF/Fc) (Fischer C. J.et al. Treatment of septic shock with tumor necrosis factor receptor.Fcfusion protein. N. Engl J. Med. 334, 1697-1702, 1996) have not only notproduced improvements but have also increased mortality due to thedisactivation of the immune system. FDA has approved two antagonistsagainst TNFα (Lipsky P E. et al. Infliximab and Methotrexate in theTreatment of Rheumatoid Arthritis. N Engl J Med, 343, 1594-1602, 2000)that have demonstrated favorable effects in the treatment of RA. One ofthese is an antibody against a subunit of the receptor for TNFα(Infliximab). The neutralization of the TNFα beyond certain levels maybring about a disactivation of the immune system as has beendemonstrated in a model with rats having RA (Colagiovanni D B. et al.TNF-alpha blockade by a dimeric TNF type I receptor molecule selectivelyinhibits adaptive immune responses. Immunopharmacol Immunotoxicol 22,627-51, 2000). A higher frequency of infections has been observed inpatients treated with Infliximab than the control groups without it(placebo groups) (Schaible T F. Long term safety of infliximab. Can JGastroenterol 14 Suppl C:29C-32C, 2000), as well as the appearance ofauto-antibodies and the development of lupus (Markham A. et al.Infliximab: a review of its use in the management of rheumatoidarthritis. Drugs 59,1341-59, 2000). Etanercept is a chimeric proteinthat is bound to the soluble receptor for TNFα and the Fc portion of theIgG1 (Moreland L W et al, Etanercept therapy in rheumatoid arthritis. Arandomized, controlled trial. Ann Intern Med 130, 478-86, 1999).Suspending the treatment produces a relapse of the patient; therefore,this drug does not cure the disease. This implies that the patientshould be treated for long periods. Although in short term studies therehave been no important adverse effects, a prolonged therapy mightgenerate the presence of antibodies against the molecule (Russell E. etal. Patients receiving etanercept may develop antibodies that interferewith monoclonal antibody laboratory assays. Arthritis Rheum 43, 944-47,2000). Several cases of patients treated with Etanercept have beenreported to have developed fatal aplastic anemia, as well aspancytopenia, and demyelating syndromes (Klippel J H, Biologic Therapyfor Rheumatoid Arthritis. N. Engl J. Med., 343, 1640-1, 2000).

Until now, no antagonist against IFNγ has been clinically used. The useof cytokine antagonists to prevent, decrease or eliminate inflammatoryand autoimmune reactions harming the body due to their temporalalteration or their chronicity, have been explored for many cytokines.Many of them have been uneffective. In some cases the antagonist islimited to a cytokine molecule and its scope of action and potency arelimited, since the doses cannot be increased because of their toxicity.In those that have been able to neutralize the cytokine, this effect hasbeen extreme and has produced a disactivation of important functions forthe body (immune system).

The IL-2 interacts with its RαIL-2. This subunit is able to internalizethe IL-2, which has been demonstrated for T and B lymphocytes and isprobably recycled to the surface, while the other chains of thisreceptor are degraded after they are internalized (Hemar A. et al.Endocytosis of interleukin 2 receptors in human T lymphocytes: distinctintracellular localization and fate of the receptor alpha, beta, andgamma chains. J Cell Biol 129, 55-64, 1995). The IFNγ is internalizedand degraded, while its receptor is recycled (Celada A. et al.Internalization and degradation of receptor-bound interferon-gamma bymurine macrophages. Demonstration of receptor recycling. J Immunol 139,147-53, 1987). The internalizing characteristics of the IL-2 by itsIL-2R α, as well as that of the IFNγ by its receptor indicates apotential recycling, at least, of the extracellular region of thereceptor for IFNγ forming part of the chimeric antagonist AnTh1. Thiswill result in a prolongation of the presence of this molecule in thebloodstream, and therefore, of its effect.

The union or conjugation of two molecules frequently leads to asignificant decrease or loss of the biological activity of the relatedmolecules because of the conformational changes occurring or to thespatial interference produced as a result of the creation of a newmolecule (Knusli, C et al. Polyethylene glycol (PEG) modification ofgranulocyte-macrophage colony stimulating factor (GM-CSF) enhancesneutrophil priming activity but not colony stimulating activity. Br JHaematol 82, 654-63, 1992). Certain alternatives have been used, as forexample in the U.S. Pat. No. 5,073,627 where there is a description of aunion peptide that fuses the granulocyte colony-stimulating factor andmacrophages (GM-CSF) with IL-3. A problem with this type of peptides isthat they can be rigid and non-flexible. As a result of this the proteincannot move to adopt the precise conformation allowing it to carry outits biological activity properly. It is therefor also necessary todesign and find peptides that may bind two molecules and at the sametime allow each one of them to adopt the proper conformation, producinga biologically active molecule. Taking the latter into account safer,more effective and more specific immuno-suppressors andanti-inflammatory drugs are needed. It is therefore important toconceive a molecule with more functional versatility, that inhibits allthe pathological activities or functions of the target drugs but that,at the same time, may allow the important functions for the body to takeplace, enabling its prolonged use without endangering the life ofpatients. An antagonist that may interfere with the simultaneous actionof two cytokines and that may avoid immuno-paralization has not yet beendesigned.

DETAILED DESCRIPTION OF THE INVENTION

The essence of this invention is that of a recombinant chimeric proteincalled AnTH1 that is formed by a 60 amino acid fragment of theN-terminal region of the human IL-2 fused through a 4 amino acid peptideto the N-terminal extracellular region of the alpha subunit of thereceptor for IFNγ containing 228 amino acids. This protein has a growthstimulating activity of T cells, it inhibits the growth stimulatingactivity of T cell induced by IL-2, it inhibits the induction of HLA-IIbγ IFNγ and it inhibits the antiproliferative activity of IFNγ. The DNAsequence corresponding to the first 60 amino acids of the N-terminal ofhuman IL-2 and the binding peptide (SEQUENCE #5) and the sequence of thebinding peptide and the DNA coding for the 228 amino acids of theextracellular region of the RαIFNγ (SEQUENCE #7) were obtained byreverse transcription of the RNA poly A from Jurkat and Raji cellsrespectively and amplified with specific primers through PCR (seeexample 1, SEQUENCE #1 and SEQUENCE #2, SEQUENCE #3, SEQUENCE #4). TheDNA sequence corresponding to the binding peptide (SEQUENCE #6) wasdesigned for that purpose and added to the primers used to amplify theregion of the soluble receptor for RαIFNγ.

The present invention also describes the vector used for production inE. coli of the recombinant protein AnTH1, called pHu (AnTH1), which wasdeposited in the Belgian Coordinated Collections of Microoganism (BCCM),with the access number LMBP 4535 on May 6, 2002. This vector wasprepared using recombinant DNA technology (Sambrook et al. MolecularCloning—A laboratory Manual, 2nd ed. Cold Spring Harbor, N.Y. 1989). Thelatter vector was modified and finally contains a marker of choice, thetryptophane promoter, and the sequence that codifies for the 60 aminoacids of human IL-2, the binding peptide and the 228 amino acids of theextracellular region of the Rα IFN γ (SEQUENCE #8). The finalconstruction of the vector obtained is called pHu (AnTH1), (see example2, FIG. 1).

The present invention also describes the transformed E. coli strain withthe pHu vector (AnTH1). The pHu vector (AnTH1) was used to transform theE. coli W3110P3 strain. The E. coli W3110P3 strain containing the pHuvector (AnTH1) was grown and induced for the expression of therecombinant chimeric protein AnTH1. The strain achieves a high degree ofexpression of the protein (see example 4). This protein may be expressedin other host cells (insect cells, mammal cells and plant cells usingthe proper vectors). The present invention also describes the productionof a recombinant chimeric protein with an adequate degree of purity forthe evaluation of its biological activity through a process by which apreparation with 80-90% purity of the biologically active protein isobtained. The cellular pellet obtained through strain culture afterinduction, was processed for the extraction of the recombinant protein.Successive washed pellet procedures were carried out using solubilizingagents (denaturalizing) that allow the extraction of the protein ofinterest with a minimum amount of contaminant proteins from E. coli.After obtaining an adequate degree of purity (80-90%) using theseprocedures, the preparation containing the AnTH 1 protein was refolded.This procedure consists of slowly eliminating the denaturalizing agents.The process is illustrated in example 5.

The invention describes the biological activity of the recombinantprotein AnTH1. This protein has a T cell growth stimulating activity(example 7). It is able to interfere with the stimulation of theproliferation of the T cells produced by the human IL-2 in the mousecell line CTLL-2 (example 8). It is able to inhibit theantiproliferative activity of the human IFNγ in the cell line Hep-2(example 9) and it also inhibits the stimulation by IFNγ of the HLA-IIexpression in COLO 205 cell line (example 10). The human IL-2, incontrast to IFNγ, can also act on murine cells.

This recombinant protein may be used in the preparation of a usefulpharmaceutical composition for diseases where both IL-2 and IFNγ have apathological role. These diseases may be of autoimmune type, such asmultiple sclerosis, sistemic lupus erythematosus, miastenia gravis,insulin dependent diabetes mellitus, active chronic hepatitis andfulminant hepatitis; of the autoimmune/inflammatory type, such asreumatoide arthritis and psoriasis, as well as in the rejection oforgans, the graft-versus-host disease and in inflammatory diseases suchas septic shock and also for atherosclerosis.

A new method is created with this invention, to prevent, interfere,and/or eliminate the actions of two cytokines that have pathogenicfunctions in different diseases. Hence, the targest are increased in amore specific form. The use of a chimeric protein that may neutralize orinterfere with two cytokine signalizing systems dealing with the samepathological situations will allow to amplify the scope of ways fortheir interference, and therefore for their efficacy. On having acertain T cell growth stimulating activity, this would avoid anundesirable inactivation of the immune system, allowing a more prolongeduse in patients and entities requiring it. Therefore, the drug would besafer and more efficient. The aim of the present invention is to createa hetero-bivalent antagonist that may interfere with the functions ofthe human IL-2 and the IFNγ, amplifying the therapeutic possibilities ofthe molecule and avoiding adverse reactions.

Microorganism Deposit:

The pHu (AnTH1) plasmid was deposited under the Treaty of Budapest forthe Protection of Microorganisms in the Belgian Coordinated Collectionsof Microoganism (BCCM), with the access number LMBP 4535 on May 6, 2002.

DESCRIPTION OF THE FIGURES

FIG. 1. Genetic construction plasmid pHu (AnTH1) for the expression ofthe recombinant chimeric protein AnTH1.

FIG. 2. Expression on E. coli of the chimeric protein AnTH1.Electrophoresis in 12.5% polyacrylamide gel under reducing conditions.a: molecular weight standar; b, c, d: negative control strain 0, 8 and18 hours of induction; e, f, g: strain under induction conditions 0, 8,18 hours containing plasmid pHu (AnTH1).

FIG. 3. Presence of the recombinant chimeric protein AnTH1 in thedifferent stages of the semipurification process. A: Electrophoresis in12.5% polyacrylamide gel under reducing conditions. In line a: molecularweight standard, b: fermentation, c: Urea 8 M extraction, d: fractionsof the mixed gel filtration chromatography, e: mixture of fractionsafter the dialysis. B: “Western blot” using and anti—AnTH1 antiserumdeluted 1:1000 in 1% PBS/non-fat milk, released from the anti-E. colistrain W3110 P3 antibodies. B: In lines a, b, c, d the same samples wereapplied and in the same order referred to in the electrophoresis ofFigure A (no molecular weight standard was applied).

FIG. 4. Identification of the chimeric protein AnTH1 using “westernblot”. Anti-IL-2 (A) and anti-AnTH1 (B) antisera were used for theidentification. The dilution factor for both was 1:1000. The AnTH1preparation refolded after dialysis was used (line a), in line b IL-2was applied.

FIG. 5. Binding assay of the human IFNγ marked with I¹²⁵ to the chimericprotein AnTH1 using “dot blot”. Approximately 1 ul of the elutedfractions from the gel filtration chromatography in nitrocellulosemembrane were used. The strips were incubated with IFNγ marked with I¹²⁵(35 μCi/μg) in the absence (line a) and in the presence (line b) of anexcess of human IFNγ (100 times more).

FIG. 6. Stimulating activity of the growth of T cells from therecombinant chimeric protein AnTH1. Bar 1: 2.8 ng/ml of IL-2, bar 2: 1.5μg/ml of AnTH1, bar 3: culture media.

FIG. 7. Inhibition by the recombinant chimeric protein AnTH1 of thegrowth stimulating activity of human IL-2 on the mouse cell line CTLL-2(results expressed in international units of IL-2).

FIG. 8. Results of the inhibition of the antiproliferative activity ofthe human IFNγ by the recombinant chimeric protein AnTH1. Bar 1: 4 IU/mlof γ IFN (4 ng/ml), bar 2: 4 IU/ml of γ IFN+50 μg/ml AnTH1, bar 3: 50μg/ml AnTH1.

FIG. 9. Inhibition of the IFNγ induced HLA II by the recombinantchimeric protein AnTH1. Bar 1: Basal level (IFNγ=0 IU/ml), bar 2: IFNγ500 IU/ml (0.5 μg/ml), bar 3: IFNγ 500 IU/ml+1.5 μg/ml AnTH1.

FIG. 10. Amino acid sequence (SEQ ID NO: 9) of recombinant chimericprotein AnTH1. Peptides defined by mass spectrometry are indicated.

EXAMPLES Example 1

Isolation of the Complementary DNA Strands that Codify for the First 60Amino Acids of the Human IL-2 and the Extracellular Region of the αSubunit of the Soluble Receptor of Human IFNγ (Rα IFN γs).

For the isolation of the human complementary DNA, Raji (Burkittlymphoma, ATCC:CCL-86) and Jurkat (human acute T cell leukemia, ATCC:TIB-152) cells were grown in the medium RPMI 1640 supplemented with 10%bovine fetal serum in a 5 L flask with gentle shaking at 37° C. in a 5%CO₂ atmosphere. The extraction of total RNA from Jurkat and Raji cellswas made through the Chomczyski method (Chomczyski P. et al. Single-stepmethod of RNA isolation by acid guanidium thiocyanate-phenol-chloroformextraction. Anal. Biochem. 162, 156-9, 1987). Then, the RNA-poly A wasextracted using a commercial messanger RNA isolation system (IsolationSystem Life Technologies Messagemakerr cat. #10551-018). Thecomplementary DNA coding for the first 60 amino acids of the human IL-2and the 288 amino acids of the extracellular amino terminal portion ofthe human Rα IFN γs were isolated by reverse transcription and amplifiedby PCR of the poly A RNA from the Jurkat and Raji cells respectively.Approximately 1-2 μg of poly A RNA was processed using random hexameres.

The sequences of the oligonucleotides (primers) used to amplify thecomplementary strand of the DNA for the first 60 amino acids of thehuman IL-2 are described in SEQUENCE #1 (primer 1) and SEQUENCE #2(reverse primer 1). The sequences of the primers used to amplify thecomplementary strand DNA for the 228 amino acids of the amino terminalportion of the human Rα IFN γs are described in SEQUENCE #3 (primer 2)and SEQUENCE # 4 (reverse primer 2).

The amplified DNA coding for the first 60 amino acids of the human IL-2and the binding peptide (SEQUENCE #5) were digested with the restrictionenzyme Nco I (underlined in the sequence of primer 1, SEQUENCE # 1).Then the extraction with phenol-chloroform was carried out to eliminatethe enzyme and the buffer. It was precipitated and resuspended in theappropriate buffer and stored at −70° C. See sequence of the bindingpeptide (SEQUENCE #6).

The amplified DNA coding for the binding peptide and the 231 amino acidsof the Rα IFNγ (SEQUENCE #7) was filled at the cohesive ends with Klenowpolimerase and purified as previously described. Then it was digestedwith the restriction enzyme BamH I (underlined in the sequence of thereverse 2 primer), and purified as described above. See sequence of thebinding peptide (SEQUENCE #6).

The amplified bands (SEQUENCES #5 and #7) were mixed in the calculatedproportions and in the appropriate buffer to obtain the amplification ofa new band containing the DNA sequence corresponding to the first 60amino acids of the human IL-2 bound to the corresponding sequence forthe 231 amino acids of the extracellular region of the human RαIFNγ andby the sequence corresponding to the binding peptide (SEQUENCE #6). Thenew band was amplified by PCR.

The amplified DNA (SEQUENCE #8) was purified as previously described.The purified DNA was digested with the restriction enzyme Bam HI andprocessed to eliminate the enzyme and the buffer, as described. Later,the DNA was again digested with the NcoI enzyme and purified asdescribed.

Example 2

Genetic Construction for the Expression of the Recombinant ChimericProtein AnTH1.

The expression vector contains the strong tryptophane promoter. Thevector was digested with the BamH I enzyme. Then the extraction wascarried out with phenol-chloroform to eliminate the enzyme and thebuffer and it was precipitated and resuspended in an appropriate buffer.Afterwards, it was digested with the Nco I enzyme. The vector wasfinally separated in a gel as previously described. Hence the vectorcontains the promoter for the tryptophane, a free cohesive site Nco I, afree cohesive site BamH I, the terminator T4 and gene for the resistanceto ampicillin. The DNA corresponding to SEQUENCE #8 was ligated to thevector using the ligase T4 enzyme. The E. coli cells were transformedwith the genetic construction. The transformants containing thecomplementary DNA fragment for the first amino acids of the human IL-2were identified, as well as the binding peptide and the 231 amino acidsof the human Rα IFN γs in the same direction as the tryptophane promoterby carrying out restriction analysis with enzymes Nco I and Eco RI. Theresulting plasmid was called pHu (AnTH1) (see FIG. 1).

Example 3

Sequencing.

The final genetic construction was sequenced. The sequencing was used inthe protocol based on the procedure of Sanger (Sanger F et al. DNAsequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA74, 5463-67, 1977). It was proven that the construction has a part ofthe genes coding for the IL-2 (60 amino acids starting from the aminoend group) under control, and later, the sequence of nucleotides codingfor the union peptide (SEQUENCE #6) bound to the region that codifiesfor the 228 amino acids of the amino end of the Rα IFN γs. See SEQUENCE#8.

Example 4

Expression in E. coli of the Recombinant Chimeric Protein AnTH1.

The host strain was Escherichia coli W3110 P3 (prototroph F-) and theplasmid pHu (AnTh1). For the expression, the plasmid was inoculated in 5mL of the LB medium with ampicillin (50 μg/mL) and L-triptophan (100μg/mL), and incubated at 37° C. for 6 hours while shaking. This culturewas added to 50 mL of the LB medium and placed in a shaker at 100 r.p.m.for 6 hours at 37° C. This culture was added to 500 mL of the M9 medium(33 mM Na₂HPO₄, 2 mM KH₂PO₄, 8.5 mM NaCl, 18 mM NH₄CL, 0.1 mM CaCl₂, 1mM MgSO₄), enriched with 0.2% hydrolized casein, 0.4% glucose, 50 μg/mLampicillin, in such a way that the initial optic density of the culture(620 nm) was of 0.3. It is then incubated for 8 hours under the sameconditions described above and finally, the cell sediment is collectedthrough centrifugation (see FIG. 2).

Example 5

Extraction, Purification and Renaturalization of the RecombinantChimeric Protein AnTH1.

The cells are homogenized with polytron at a concentration of 0.1 g ofthe damp biomass per mL of the TE buffer (10 mM Tris HCl, 1 mM. EDTA pH7.2). This suspension is submitted to an enzymatic rupture process withlysozime. The pellet obtained in the previous step is submitted to cellwashing with different molarities of urea, from 1M to 8M, in 50 mM ofTris pH 7.2, 1 mM of EDTA. The homogenization is carried out withpolytron. Initially it is homogenized for 1 minute, left to rest for 3minutes and then again homogenized another minute. The whole process iscarried out at 4° C. The proteins that are solubilized with urea,approximately 150 mL, were applied on a flow of 3 mL/minute on a K9/60column (Pharmacia, Sweden), containing Sephadex G-100 resin that waspreviously equilibrated with 3 volumes of 50 mM of Tris HCL pH 9, 4Murea. The elution was carried out in the same buffer. The fractionscontaining the proteins were combined and dialyzed against 0.1 M of TrisHCl pH 9. Afterwards, the dialysis continued against the phosphatebuffer saline (PBS) pH 7.4. (see FIG. 3 y 4)

Example 6

Binding of Gamma IFN to the Recombinant Chimeric Protein AnTh1.

One microliter was applied (approximately 1 μg of total protein) of therecombinant chimeric protein AnTH1 after folding to nitrocellulosestrips. The strips were incubated with 10% non-fat milk for 2 hours atroom temperature (RT). The membranes were washed twice with Tris Buffersaline (TBS) for 5 minutes. After washing, the strips were incubatedwith gamma IFN marked with radioactive iodine (¹²⁵I-IFN gamma) 35 μci/μgfor 1 hour at RT in the presence or absence of an excess of gamma IFNthat was not marked. Afterwards, the nitrocellulose strips were washedtwice with TBS for 5 minutes and then with TBS+0.03% Tween 20 for 5minutes. Finally, radiographic films were exposed to the strips andstored at −70° C. for 72 h and then developed. (See FIG. 5).

Example 7

Stimulating Activity of the Growth of T Cells of the RecombinantChimeric Protein AnTH1.

The biological activity of the recombinant chimeric protein AnTH1 wastested using the cell line of murine T lymphocytes dependent on IL-2.The cells were grown on the medium RPMI-1640 containing 1 mM pyruvate, 2mM L-glutamine, 40 mM HEPES, 100 U/mL of penicillin, 50 μg/mLstreptomycin, 50 μM 2-mercaptoethanol and 10% bovine fetal serumsupplemented with 8 IU/mL of human recombinant interleukin-2 with aspecific activity of 1.2×10⁷ IU/mg. Before using the cells are washed 3times, resuspended in the complete culture medium without IL-2 andincubated for 1 hour at 37° C. in a damp CO₂ atmosphere. Then the cellswere washed, resuspended at a density of 4×10⁵ cells/mL and distributedin plates of 96 wells (100 μl per well) containing 100 μl of serialdilutions 1:2 of rhIL-2 or samples (the recombinant chimeric proteinAnTH1), in a complete medium. The international standard used in thistrial was IL-2010397. After 36 hours of incubation at 37° C., 20 μl of 5mg/mL of MTT (C₁₈H₁₆N₅SBr) were added to each well and the plates wereincubated for 4 hours under the same conditions. Finally, 50 μl/pozo ofthe 10% SDS, 0.1 N HCl, 50% isopropanol were added, the plates wereshaken for 1 hour at 37° C., and the absorbance was read at 570 nm usingthe plate reader. (See FIG. 6).

Example 8

Inhibition of the IL-2 Activity of Stimulating the Growth of T Cellswith the Recombinant Chimeric Protein AnTH1.

The biological activity for IL-2 was tested using the cell line ofmurine lymphocyte T dependent on IL-2. The cells were grown in a similarform as the previous experiment. Before they were used the cells werewashed 3 times, resuspended in the complete culture medium without IL-2and incubated for 1 hour at 37° C. in a humid CO₂ atmosphere. Then thecells were washed, resuspended at a density of 4×10⁵ cells/mL anddistributed in 96-well plates (100 μl per well) containing 100 μl of theserial dilutions 1:2 of rhIL-2 or samples (rhIL-2+the recombinantchimeric protein AnTH1 or the recombinant chimeric protein AnTH1 alone),in a complete medium. The international standard used in this trial wasIL-2010397. After 36 hours of incubation at 37° C., 20 μl of 5 mg/mL ofMTT (C₁₈H₁₆N₅SBr) were added to each well and the plates were incubatedfor 4 hours under the same conditions. Finally, 50 μl/well of thesolution of 10% SDS, 0.1 N HCl, 50% isopropanol were added. Then theplates were shaken for 1 hour at 37° C., and the absorbance was read at570 nm using a plate reader. The results were expressed as units ofrhIL-2, based on the analyses of the data of the standard dilution curveof rhIL-2 and the serial dilutions of the sample (See FIG. 7).

Example 9

Inhibition of the Antiproliferative Activity of the γ IFN by theRecombinant Chimeric Protein AnTH1.

The growth of 2.5×10³ cells/well of Hep-2 cultured in a MEM CANE medium(essential minimum medium with non-essential amino acids) supplementedwith 10% bovine fetal serum took place in 96 well plates. They wereincubated for 24 hours at 37° C. in an incubator with 5% of CO₂. Afterthis time the medium was changed and the samples to be evaluated wereadded in serial dilutions as well as their respective controls. After 72hours of incubation the cells were stained with 0.5% crystal violet for2 minutes and the plates were read in a plate reader. (See FIG. 8).

Example 10

Inhibition of the induction bγ IFN γ of HLA II by the recombinantchimeric protein AnTH1.

This is an ELISA trial on cells described by Seeling G. et al.Development of receptor peptide antagonist to human γ-interferon andcharacterization of its ligand-bound conformation using transferrednuclear overhauser effect spectroscopy. J. Biol. Chem. 270, 9241-53,1995. A cell line Colo 205 was used. They were grown in culture plateswith 96 wells, 2.5×10⁵ cells/well in 0.1 mL of RPMI 1640 enriched with10% bovine fetal serum. The cells were incubated for 12 hours at 37° C.in an incubator with a 5% CO₂ atmosphere. Then the cells grown in theculture medium were added in the presence of the recombinant chimericprotein and of γ IFN in a volume of 0.1 mL and then incubated for 1 hourat 37° C. After incubation, the medium was removed and the wells werewashed 3 times with the culture medium. Afterwards, aliquots of 1.2 mLof the medium were added to the wells and the plates were incubated for48 hours at 37° C. to allow for the induction of the HLA-DR antigens.The wells were washed with PBS and the cells were fixed with pureethanol for 2 minutes. Washing was repeated and the plates wereincubated for 1 hour at room temperature with a mouse monoclonalantibody anti-HLA-DR diluted in PBS 0.5% of bovine serum albumin. Thewells were washed with PBS and incubated under the same conditions withthe conjugate anti-IgG mouse-peroxidase. The washings were repeated 3times and developed by adding 100 μL/well of 0.15% H₂O₂+5 mg/mLo-phenylendiamine. The detection of the reaction was performed with 50μL/well of 2 M H₂SO₄ and absorbance was measured at 492 nm in the platereader. (FIG. 9).

Example 11

Mass Spectrometric Analysis

An aliquot (0.5 μg) of the purified protein was analyzed by SDS-PAGE andreversed-stained with Zn-immidazol (Castellanos-Serra L, y cols.Detection of biomolecules in electrophoresis gels with salts ofimidazole and zinc II. a decade of research. Electrophoresis. 2001, 22,864-7). The band was excised and incubated with a citric acid solution(1%) during 5 minutes until complete colorless and incubated another 10minutes in water to remove the excess of chelating agent. Thetransparent band was additionally cut in small cubes of 1 mm³approximately, and dehydrated in a 90% acetonitrile aqueous solutionwith no TFA and completely dried in speed-vac. The gels pieces wererehydrated in 20-30 μL of a 50 mM NH₄HCO₃ solution containing 12.5 ng ofa modified trypsin, sequencing grade from Promega (MA, USA). The in-geldigestion was incubated overnight at 37° C. in a termomixer (Eppendorf,USA). Additional 20 μL of 50 mM NH₄HCO₃ solution were added andadditional 45 min were incubated and the tryptic peptides were extractedby using ZipTips C18 from Millipore (USA) previously activated andequilibrated as recommended by the manufacturer. Twenty loading cycleswere carried out for extracting the tryptic peptides. The digest wasacidified with formic acid, incubated 45 minutes at room temperature andanother twenty loading cycles were achieved. The Ziptips were washedextensively by using a 5% formic acid solution and the proteolyticpeptides were eluted in 2 μL of 60% acetonitrile containing 1% of formicacid.

The low-energy MS/MS spectra were acquired using a hybrid quadrupoleorthogonal acceleration tandem mass spectrometer QTOF from Micromass(Manchester, UK) fitted with a Z-spray nanoflow electrospray ion source.The mass spectrometer was operated with a source at 80° C. and a dryinggas flow of 50 L/h. Peptides were dissolved to reach an approximateconcentration of 5 pmole/μL. Two microliters of the tryptic peptideswere loaded onto the borosilicate nanoflow tip and 900 V and 35 Vpotential were applied to the nanoflow tip and entrance cone,respectively. To acquire the MS/MS spectra was used the method describedby González, L. y cols. Differentiating alpha- and beta-aspartic acidsby electrospray ionization and low-energy tandem mass spectrometry.Rapid. Commun. Mass Spectrom. 2000, 14, 2092-210. The first quadrupolewas used to select the precursor ion within a window of 4-5 Th. Apressure of ˜3×10⁻² Pa collision gas (argon) was used in the hexapolecollision cell to yield the fragment ions. Appropriate collision energywas used to reduce the intensity of the precursor ion to more than halfof its original intensity. Data acquisition and processing wereperformed using a MassLynx system (v 3.5) from Micromass.

ESI-MS analysis of the tryptic peptides derived from the AnTh1 protein #Peptidic sequence ^(a)) m/z exp. m/z teor. Z ^(b)) Abs. Error ^(c)) 1¹¹T-K^(33 d)) 908.8 908.83 3 0.02 2 ⁴⁰M-K⁴⁴ 639.3 639.35 1 0.01 3⁴⁵F-K⁵⁰ 685.3 685.33 1 0.00 4 ⁵⁶H-R^(66 f)) 442.8 442.88 3 0.01 5⁶⁷A-K¹¹⁴ 1781. 1781.54 3 0.02 6 ¹⁰¹Q-K^(114 d), e)) 807.9 807.95 2 0.027 ¹²⁰N-R^(151 f)) 921.1 921.17 4 0.03 8 ¹⁶⁶S-R¹⁷³ 506.2 506.23 2 0.00 9¹⁷⁷I-K¹⁸¹ 511.3 511.28 1 0.03 10 ¹⁹⁰Q-R^(220 f)) 1225. 1225.23 3 0.03 11²²¹V-R^(227 d)) 456.7 456.75 2 0.01 12 ²²⁸M-K^(236 d)) 535.2 535.25 20.01 ^(a)) The numbering in the sequences of peptides is according tothe AnTh1 protein shown in FIG. 10 (SEQ ID NO:9) ^(b)) Charge state ofthe individual peptides. ^(c)) Indicate the absolute mass differencebetween the theoretical and experimental molecular masses of thedetected peptides. ^(d)) Peptides sequenced by ESI-MS/MS. ^(e)) Peptideoriginated by the non-specific cleavage of trypsin. ^(f)) Peptidecontaining free cysteineAdvantages of the Proposed Solution.

The invention combines in one molecule the ability to intervene in twosignaliation systems affecting immuno-regulating and inflammatorymechanisms. The design of the chimeric protein consists of the fusion ofa ligand (IL-2₆₀) through a 4 amino acid peptide with an extra-cellularreceptor (Rα IFN γ). This combination allows the binding of therecombinant chimeric protein with the cells containing on their surfacethe Rα IL-2. This subunit is mainly present in non-activated T cells andin the high affinity IL-2 receptor (Rαβγ IL-2) in the activated T cells(Smith, K. A. The interleukin-2 receptor. Annu. Rev. Cell. Biol. 5,397-403, 1989 and Strom, T. B. et al. Interleukin-2 receptor-directedtherapies: antibody-or cytokine-based targeting molecules. Annu. Rev.Med. 44, 343-50, 1993).

If the AnTH1 protein binds to the RαIL-2 in the cells at rest, it caninternalize the protein, leaving Rα IFN γs in the cytoplasm under theconditions of being recycled outwards and interfering with the γ IFNthat will be produced on activating the cells. The interaction of the γIFN from the cytoplasm with an intracellular region of the membranereceptor that may generate the biological activity of γ IFN, has beendescribed (Szente B. E. et al. Identification of IFN (receptor bindingsites for JAK2 and enhancement of binding by IFN γ and its C-terminalpeptide IFN γ (95-133). J. Immunol. 155, 5617-22, 1995). During adisease where there may be a need of decreasing the action of the IL-2produced by the body when adding the AnTH1 protein, this will bindthrough the IL-2₆₀ portion to the alpha subunit in the high affinitycomplex (Rαβγ IL-2) in the activated cells, it will interfere with thebinding of the complete IL-2 (native) secreted by the cells of theimmune system and will interfere in its biological activity. On theother hand the γ IFN that has already been secreted by the activated Tcells can be sequestered by the Rα IFN γs portion of the chimericprotein, avoiding its attachment to the membrane receptor. In this way,an autoimmune, and/or inflammatory reaction can be controlled in twodifferent moments, during the activation and in the process ofpropagating the reaction.

The invention offers a hetero-bivalent chimeric protein that mayinterfere with the biological activity of the IL-2 and the γ IFN.Considering that the recombinant chimeric protein AnTH1 also has a Tcell growth stimulating activity, a less profound inactivation of theimmune system may be expected, which will provoke antagonistanticytokines that were already clinically proven.

1. A recombinant chimeric protein comprising the amino acid sequence setforth in SEQ ID NO:
 9. 2. A pharmaceutical composition comprising aneffective amount of a recombinant chimeric protein, wherein the proteincomprises the amino acid sequence set forth in SEQ ID NO: 9.